Mutational and Nonmutational Activation of p21 in Rat Colonic Azoxymethane- induced Tumors: Effects on Mitogen-activated Protein Kinase, Cyclooxygenase-2, and Cyclin D1

Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for b-catenin. Prior in vitro studies have also demonstrated that wild type p21 can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21 activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21 activation levels (8.8 6 1.5%; n 5 13) compared with that of control colon (3.7 6 0.4; n 5 6; P < 0.05) or tumors without such mutations (4.2 6 0.4%; n 5 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21 activation levels (8.0 6 0.9%; n 5 18) compared with control colons. In three of four tumors examined with activated wild-type p21, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21 demonstrated these alterations. Mitogenactivated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21, compared with their nonactivated counterparts. Although b-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or b-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or b-catenin mutations or nonmutational activation of p21.